Project Summary/Abstract DNA replication is essential to maintain the genome of all organisms. During each round of cell division, eukaryotic cells must establish hundreds to thousands of replication forks that coordinately replicate each chromosome. The eukaryotic replicative DNA helicase, the Mcm2-7 complex, is at the core of these events. During G1, loading of this ring-shaped protein around DNA marks all potential origins of DNA replication. Upon entry into S phase, these helicases are activated in a process that dramatically remodels both the helicases and their associated DNA. Importantly, it is the temporal separation of these two events that ensures that no sequence is replicated more than once per cell cycle. Consistent with their importance, defects in or misregulation of replication initiation is known to lead to cancer and developmental abnormalities. Thus, understanding the mechanism of these processes will provide critical information concerning the maintenance of genome integrity. The biochemical reconstitution of the events of replication initiation has provided powerful tools to understand these events. These ensemble assays have identified a relative order of initial protein binding to the origin DNA and the requirements of each protein for a few detectable events (e.g. DNA unwinding or synthesis). On the other hand, these assays are poorly suited to study the complex protein dynamics involved in replication initiation due to their incomplete and asynchronous nature. Single-molecule biochemical studies bypass these problems by monitoring events on individual DNA molecules in real time. Further development of single-molecule assays for the events of replication initiation will provide critical insights into dynamic events of DNA replication and complement the growing abundance of static structures of DNA replication proteins/complexes. The proposed research focuses on extending the use of single-molecule assays to investigate eukaryotic replication initiation. Specific aim one expands on existing single-molecule assays of helicase loading to address three key questions: (1) When and how is the Mcm2-7 ring opened and closed during helicase loading?; (2) How is recruitment of the second Mcm2-7 distinct from the first?; and (3) What events in helicase loading are catalyzed by Mcm2-7 ATP hydrolysis? The second aim proposes the development of single-molecule assays for helicase activation and their use in understanding the coordinated activation of both helicases at the origin. The last aim addresses the mechanisms that remodel the initially loaded helicase dimer into two monomers that encircle single- stranded DNA.